Comparative genome analysis of Sesbania cannabina-nodulating Rhizobium spp. revealing the symbiotic and transferrable characteristics of symbiosis plasmids

Abstract

Symbiotic nitrogen fixation between legumes and rhizobia makes a great contribution to the terrestrial ecosystem. The successful symbiosis between the partners mainly depends on the nod and nif genes in rhizobia, while the specific symbiosis is mainly determined by the structure of Nod factors and the corresponding secretion systems (type III secretion system; T3SS), etc. These symbiosis genes are usually located on symbiotic plasmids or a chromosomal symbiotic island, both could be transferred interspecies. In our previous studies, Sesbania cannabina-nodulating rhizobia across the world were classified into 16 species of four genera and all the strains, especially those of Rhizobium spp., harboured extraordinarily highly conserved symbiosis genes, suggesting that horizontal transfer of symbiosis genes might have happened among them. In order to learn the genomic basis of diversification of rhizobia under the selection of host specificity, we performed this study to compare the complete genome sequences of four Rhizobium strains associated with S. cannabina, YTUBH007, YTUZZ027, YTUHZ044 and YTUHZ045. Their complete genomes were sequenced and assembled at the replicon level. Each strain represents a different species according to the average nucleotide identity (ANI) values calculated using the whole-genome sequences; furthermore, except for YTUBH007, which was classified as Rhizobium binae, the remaining three strains were identified as new candidate species. A single symbiotic plasmid sized 345-402 kb containing complete nod, nif, fix, T3SS and conjugal transfer genes was detected in each strain. The high ANI and amino acid identity (AAI) values, as well as the close phylogenetic relationships among the entire symbiotic plasmid sequences, indicate that they have the same origin and the entire plasmid has been transferred among different Rhizobium species. These results indicate that S. cannabina stringently selects a certain symbiosis gene background of the rhizobia for nodulation, which might have forced the symbiosis genes to transfer from some introduced rhizobia to the related native or local-condition-adapted bacteria. The existence of almost complete conjugal transfer related elements, but not the gene virD, indicated that the self-transfer of the symbiotic plasmid in these rhizobial strains may be realized via a virD-independent pathway or through another unidentified gene. This study provides insight for the better understanding of high-frequency symbiotic plasmid transfer, host-specific nodulation and the host shift for rhizobia.

Keywords: Rhizobium; Sesbania cannabina; conjugal transfer; secretion system; symbiotic plasmid.

Fig. 1.

The complete genome and symbiotic plasmid sizes of S. cannabina-nodulating rhizobia.

Fig. 2.

Maximum-likelihood phylogenetic tree based on the whole-genome sequences (a) and the symbiotic plasmid (pSym) sequences (b). The trees were reconstructed using iq-tree based upon the common shared single copy orthologue genes of the complete genome (a) and the pSyms (b). Bootstrap support values were calculated from 1000 replicates，scale bar units are number of substitutions per site.

Fig. 3.

The heatmap of ANI and AAI values between the S. cannabina-nodulating rhizobia and the reference strains. The ANI heatmap generated by the complete genome sequences (a) and symbiotic plasmid (c); the AAI heatmap generated by the complete genome (b) and symbiotic plasmids (d).

Fig. 4.

Homology analysis for symbiotic plasmids of S. cannabina-nodulating rhizobia. The diagram shows the number of common shared core genes, the numbers of genes shared by each two strains and the strain-specific genes for each strain.

Fig. 5.

Genetic organization of symbiotic nitrogen fixation and conjugal transfer related genes in the symbiotic plasmid. (a) Nod factor synthesis genes; (b) nitrogen-fixation-related genes; (c) T3SS genes; (d) conjugal transfer related genes.